Extensively hydrolysed infant formula

Abstract

To provide an extensively hydrolysed infant formula containing a reduced amount of protein.SOLUTION: An extensively hydrolysed infant formula (eHF) comprises protein, carbohydrate, and fat, the eHF comprising about 2.4 g or less of protein per 100 kcal, with about 30 wt.% or less of the fat being medium chain triglycerides (MCTs), and optionally further comprising 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an extensively hydrolyzed infant formula (eHF) containing reduced amounts of protein. [Background technology]

[0002] Cow's milk protein (CMP) is a leading cause of food allergy in infants, affecting 2-3% of children worldwide. Most children with CMP allergy (CMPA) have two or more symptoms: 50-70% have skin symptoms; 50-60% have gastrointestinal symptoms; and 20-30% have respiratory tract symptoms. 10% of children may develop severe, life-threatening symptoms. Management of these patients focuses on avoidance of CMP and prompt recognition and treatment of allergic reactions following accidental exposure (Nutten, 2018. EMJ Allergy Immunol, 3(1), pp. 50-59).

[0003] Human breast milk and breastfeeding are considered the optimal form of nutrition for healthy infants in the first few months of life. Breast milk remains the gold standard for feeding infants with CMPA. The European Society for Pediatric Gastroenterology and Hepatology (ESPGHAN) recommends that the complete elimination of cow's milk from the mother's diet is the best treatment for CMPA in breastfed infants (Koletzko, S., et al., 2012. Journal of pediatric gastroenterology and nutrition, 55(2), pp. 221-229).

[0004] If breastfeeding is not possible, specialized infant formulas are recommended. ESPGHAN recommends that non-breastfed infants with CMPA use extensively hydrolyzed protein-based formulas, which have been proven effective in infants with CMPA. In infants with very severe or life-threatening symptoms, amino acid formulas may be considered as the first choice (Koletzko, S., et al., 2012. Journal of pediatric gastroenterology and nutrition, 55(2), pp. 221-229).

[0005] Extensively hydrolyzed infant formulas (eHFs) may have lower nitrogen absorption rates than complete protein formulas or human breast milk (Rigo, J., et al., 1995. European Journal of Clinical Nutrition, 49, pp. S26-38). Therefore, to meet the needs of infants, eHFs typically contain 2.6-2.8g of protein per 100kcal (Borschel, M., et al., 2018. Nutrients, 10(3), p. 289).

[0006] However, consumption of high-protein infant formula has been associated with increased body weight and body mass index at age 2 years, as well as increased circulating concentrations of plasma essential amino acids, insulin-like growth factor-1, and C-peptide, which may induce weight gain and lipogenic activity. Lower protein content may reduce the risk of subsequent obesity (Totzauer, M., et al., 2018. Obesity, 26(7), pp. 1203-1210).

[0007] Therefore, there is a need for eHF with reduced amounts of protein that supports proper growth and development in allergic infants and is safe and well tolerated.

[0008] [Summary of the Invention] The inventors have developed eHF with reduced amounts of protein, and thus the eHF described herein has a protein content closer to that of human breast milk and may reduce the risk of obesity later in life.

[0009] The inventors surprisingly showed that eHF supported proper growth and development in allergic infants. Furthermore, eHF was safe and well tolerated. This may be due, at least in part, to the degree of hydrolysis, the very low amount of peptides >1200 Da, and / or the absence of medium-chain triglycerides (MCTs).

[0010] The inventors surprisingly showed that eHF supported proper growth and development in allergic infants. Furthermore, eHF was safe and well tolerated. This may be due, at least in part, to the degree of hydrolysis, the very low amount of peptides >1200 Da, and / or the low level / absence of medium-chain triglycerides (MCTs) and / or the presence of human milk oligosaccharides (HMOs).

[0011] Thus, in one aspect, the present invention provides an extensively hydrolyzed infant formula (eHF) comprising protein, carbohydrate, and lipid, wherein the eHF comprises about 2.4 g or less of protein per 100 kcal, and about 30% or less by weight of the lipid is medium chain triglycerides (MCTs).

[0012] The eHF may preferably contain one or more human milk oligosaccharides (HMOs). In one embodiment, the eHF contains 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT), preferably 2'FL and LNnT. The eHF may contain 0.5 to 3 g / L, 0.8 to 1.5 g / L, or about 1 g / L of 2'FL. Preferably, the eHF contains about 1 g / L of 2'FL. The eHF may contain 0.2 to 1 g / L, 0.5 to 0.8 g / L, or about 0.5 g / L of LNnT. Preferably, the eHF contains about 0.5 g / L of LNnT. More preferably, the eHF contains about 1 g / L of 2'FL and about 0.5 g / L of LNnT.

[0013] The eHF may contain about 1.8 g or more of protein per 100 kcal. For example, the eHF may contain 1.8 to 2.4 g of protein per 100 kcal, 2.1 to 2.3 g of protein per 100 kcal, or 2.15 to 2.25 g of protein per 100 kcal. Preferably, the eHF contains about 2.2 g of protein per 100 kcal.

[0014] About 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, 5% by weight or less, or 1% by weight or less of the lipids can be medium chain triglycerides (MCTs). Preferably, about 0% by weight of the lipids are MCTs and / or the eHF does not contain added MCTs. Most preferably, the eHF does not contain added MCTs.

[0015] At least about 95%, at least about 98%, at least about 99%, or about 100% by weight of the peptides in the eHF may have a molecular weight of less than about 3000 Da. Preferably, there are no detectable peptides in the eHF greater than or equal to about 3000 Da in size.

[0016] At least about 90%, at least about 95%, at least about 98%, or at least about 99% by weight of the peptides in the eHF can have a molecular weight of less than about 1500 Da. Preferably, at least about 99% of the peptides in the eHF have a molecular weight of less than about 1500 Da.

[0017] At least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% by weight of the peptides in the eHF can have a molecular weight of less than about 1200 Da. Preferably, at least 98% by weight of the peptides have a molecular weight of less than about 1200 Da.

[0018] At least about 45 wt.%, at least about 50 wt.%, 45-55 wt.%, or 50-54 wt.% of the peptides in the eHF can be dipeptides and tripeptides. Preferably, about 51-53 wt.%, or more preferably about 52 wt.%, of the peptides in the eHF are dipeptides and tripeptides.

[0019] At least about 45%, at least about 50%, 45-55%, or 50-54% by weight of the peptides in the eHF can have a molecular weight of 240-600 Da. Preferably, about 51-53%, or more preferably about 52% by weight of the peptides in the eHF have a molecular weight of 240-600 Da.

[0020] At least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the protein can be whey protein. Preferably, the source of the protein is whey protein.

[0021] The eHF may contain free amino acids. The free amino acids may be present at a concentration of 50% by weight or less, 40% by weight or less, 30% by weight or less, or 25% by weight or less, based on the total weight of the amino acids. Preferably, the free amino acids are present at a concentration of 20-25% by weight, 21-23% by weight, or about 22% by weight, based on the total weight of the amino acids.

[0022] The eHF may contain 9-14 g of carbohydrates per 100 kcal and / or 4.0-6.0 g of fat per 100 kcal. The eHF may have an energy density of 60-72 kcal per 100 mL.

[0023] In another aspect, the present invention provides a method of feeding an infant, the method comprising administering to the infant an eHF according to the present invention. Preferably, the infant has a cow's milk protein allergy.

[0024] In another aspect, the present invention provides an eHF according to the present invention for use in the treatment of cow's milk protein allergy, chronic diarrhea, and / or malabsorption. In one embodiment, the present invention provides an eHF according to the present invention for use in the treatment of cow's milk protein allergy.

[0025] In another aspect, the present invention provides an eHF according to the present invention for use in preventing and / or reducing the risk of obesity.

[0026] In another aspect, the present invention provides a method for preparing eHF according to the present invention. [Brief explanation of the drawings]

[0027] [Figure 1] Weight Mean weight (in kg) versus months from baseline visit for test formula (low protein + HMO) and control formula. There were no significant differences between groups at any time point. [Figure 2] Weight-for-age Z-scores Mean weight-for-age Z-scores versus months from baseline visit for the test formula (low protein + HMO) and control formula. There were no significant differences between groups at any time point. [Figure 3] Non-inferiority: Daily weight gain Treatment effect on weight gain at Visit 4. The primary analysis shows that weight gain [g / d] of infants fed the test formula was non-inferior to growth on the control formula. [Figure 4] Height-for-age Z-scores Mean height-for-age Z-scores versus months from baseline visit for the test formula (low protein + HMO) and control formula. There were no significant differences between groups at any time point. [Figure 5]Head Circumference Z-Scores for Age Mean head circumference Z-scores for age versus months from baseline visit for test formula (low protein + HMO) and control formula. There were no significant differences between groups at any time point. [Figure 6] BMI-for-age Z-scores Mean BMI-for-age Z-scores versus months since baseline visit for test formula (low protein + HMO) and control formula. There were no significant differences between groups at any time point. DETAILED DESCRIPTION OF THE INVENTION

[0028] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0029] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0030] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including" or "includes" or "containing" or "contains" and are inclusive, i.e., open-ended, and do not exclude additional, unrecited components, elements, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0031] As used herein, the term "about" means approximately, in the region of, roughly, or in the vicinity of. When the term "about" is used in conjunction with a numerical value or range, that value or range modifies that value or range by extending the boundaries above and below the stated numerical value(s). In general, the terms "about" and "approximately" are used herein to adjust numerical value(s) above and below the stated value(s) by 10%.

[0032] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Any reference cited herein should not be construed as an admission that such reference constitutes prior art to the claims appended hereto.

[0033] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numerical ranges are inclusive of the numbers defining the range.

[0034] Highly Hydrolyzed Formula The term "extensively hydrolyzed infant formula" or "eHF" can refer to an infant formula that contains extensively hydrolyzed proteins. An eHF can be a hypoallergenic infant formula that provides complete nutrition for infants who cannot digest intact CMP or who have an intolerance or allergy to CMP.

[0035] The term "infant formula" may refer to a food for a specific nutritional purpose for infants during the first year of life, which formula itself meets the nutritional requirements of humans falling within said category (as defined in Commission Regulation (EU) 2016 / 127 of 25 September 2015).

[0036] A "hypoallergenic" composition is one that is unlikely to cause an allergic reaction. Preferably, the eHF of the present invention is tolerated by more than 90% of infants with CMPA. This is in line with the guidance provided by the American Academy of Pediatrics (Committee on Nutrition, 2000. Pediatrics, 106(2), pp. 346-349). Preferably, the eHF of the present invention may not contain peptides that are recognized by CMP-specific IgE, for example, in subjects with CMPA.

[0037] Infants can be fed eHF alone, or eHF can be used as a supplement to human breast milk.

[0038] The eHF of the present invention can be in powder or liquid form.

[0039] The liquid may be, for example, concentrated liquid eHF or ready-to-feed eHF. The eHF may also be in the form of reconstituted eHF (i.e., liquid eHF reconstituted from a powder form). Concentrated liquid eHF can preferably be diluted, for example, by adding water, to a liquid composition suitable for feeding to an infant.

[0040] In one embodiment, the eHF is in powder form, which can be reconstituted, for example, by adding water, into a liquid composition suitable for feeding to an infant.

[0041] When formulated as directed, eHF may have an energy density of about 60-72 kcal per 100 mL. Preferably, when formulated as directed, eHF may have an energy density of about 60-70 kcal per 100 mL.

[0042] protein The term "protein" includes peptides and free amino acids. The protein content of eHF can be calculated by any method known to those skilled in the art. Preferably, the protein content can be calculated by the nitrogen protein equivalent method, as described, for example, in Maubois, J.L. and Lorient, D., 2016. Dairy science & technology, 96(1), pp. 15-25. Preferably, the protein content is calculated as the nitrogen content x 6.25, as defined in European Commission Regulation (EU) 2016 / 127 of 25 September 2015. The nitrogen content can be determined by any method known to those skilled in the art. For example, the nitrogen content can be measured by the Kjeldahl method.

[0043] Protein concentration eHF typically contains 2.6-2.8g of protein per 100kcal to meet the needs of infants with gastrointestinal pathologies, including severe malabsorption, or who require more protein and calories to compensate for a high metabolic rate.

[0044] The inventors have surprisingly shown that eHF with a lower protein content can support proper growth and development in allergic infants. Furthermore, the inventors have surprisingly shown that this eHF is safe and well tolerated.

[0045] Thus, an eHF of the present invention contains about 2.4 g or less of protein per 100 kcal. For example, an eHF of the present invention can contain about 2.3 g or less of protein per 100 kcal, 2.25 g or less of protein per 100 kcal, or 2.2 g or less of protein per 100 kcal.

[0046] Preferably, the eHF contains about 1.8 g or more of protein per 100 kcal. For example, the eHF of the present invention may contain about 1.86 g or more of protein per 100 kcal, 1.9 g or more of protein per 100 kcal, 2.0 g or more of protein per 100 kcal, or 2.1 g or more of protein per 100 kcal. Preferably, the eHF contains about 1.86 g or more of protein per 100 kcal, in line with current EU regulations (EFSA NDA Panel, 2014. EFSA journal, 12(7), 3760).

[0047] The eHF of the present invention may contain 1.8 to 2.4 g of protein per 100 kcal, 1.86 to 2.4 g of protein per 100 kcal, 1.9 to 2.4 g of protein per 100 kcal, 2.0 to 2.4 g of protein per 100 kcal, 2.0 to 2.3 g of protein per 100 kcal, 2.1 to 2.3 g of protein per 100 kcal, or 2.15 to 2.25 g of protein.

[0048] Preferably, the eHF contains about 2.2 g of protein per 100 kcal.

[0049] Protein Source The source of protein can be any source suitable for use in infant formula. Preferably, the protein is cow's milk protein.

[0050] The highly hydrolyzed / hydrolyzed whey-based formula may be more palatable than the highly hydrolyzed / hydrolyzed casein-based formula, and / or the subject may be only sensitive to casein protein. Therefore, preferably, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or about 100% of the protein is whey protein. Preferably, the source of the protein is whey protein.

[0051] The whey protein may be whey derived from cheese production, in particular sweet whey such as that obtained by coagulation of casein with rennet, acid whey obtained by coagulation of casein with acid or by acidification of a ferment, or even mixed whey obtained by coagulation with acid and with rennet. The raw material may be whey that has been desalted by ion exchange and / or by electrodialysis, known as desalted whey protein (DWP).

[0052] The source of whey protein can be sweet whey from which caseinoglycomacropeptide (CGMP) has been completely or partially removed. This whey is called denatured sweet whey (MSW). Removal of CGMP from sweet whey results in a protein material with a threonine and tryptophan content similar to that of human breast milk. The process for removing CGMP from sweet whey is described in EP 880902.

[0053] The whey protein may be a mixture of DWP and MSW.

[0054] In some embodiments, the amount of casein in the eHF is undetectable, e.g., less than 0.2 mg / kg. The amount of casein can be determined by any method known to one of skill in the art.

[0055] Degree of hydrolysis In eHF, proteins are "extremely hydrolyzed," so that eHF can be tolerated by more than 90% of infants with CMPA.

[0056] The protein hydrolysate may have a degree of hydrolysis characterized by NPN / TN%, which refers to non-protein nitrogen divided by total nitrogen x 100. Non-protein nitrogen refers to amino nitrogen that is free to react with reagents such as trinitrobenzenesulfonic acid (TNBS). NPN / TN% may be determined by any method known to those skilled in the art. For example, NPN / TN% may be measured as described in Adler-Nissen (Adler-Nissen, J. (1979) J. Agric. Food Chem. 27:1256-1262). Preferably, the protein may have an NPN / TN% of greater than 90%, greater than 95%, or greater than 98%.

[0057] The degree of hydrolysis can also be determined by the degree of hydrolysis. The "degree of hydrolysis" (DH) is defined as the percentage of broken peptide bonds in a protein hydrolysate and can be determined by any method known to those skilled in the art. Preferably, the degree of hydrolysis is determined by pH stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), or formol titration. (Rutherfurd, SM, 2010. Journal of AOAC International, 93(5), pp. 1515-1522). The degree of hydrolysis (DH) of a protein can be greater than 90, greater than 95, or greater than 98.

[0058] The degree of hydrolysis can also be determined by peptide molecular weight distribution. Peptide molecular weight distribution can be determined by high-performance size exclusion chromatography (HPSEC / UV), optionally using a UV detector (Johns, PW, et al., 2011. Food Chemistry, 125(3), pp. 1041-1050). For example, peptide molecular weight distribution can be an estimate based on HPSEC peak area determined at 205 nm, 214 nm, or 220 nm. Preferably, when peptide molecular weight distribution is determined by HPSEC / UV, the "weight percent of peptides" having a specific molecular weight can be estimated by the "peak area fraction as a percentage of the total peak area" having a molecular weight determined at 205 nm, 214 nm, or 220 nm. Preferably, the degree of hydrolysis can be determined by the method described in WO 2016 / 156077. Alternatively, the peptide molecular weight distribution can be determined by any method known to those skilled in the art, for example, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Chauveau, A., et al., 2016. Pediatric Allergy and Immunology, 27(5), pp. 541-543).

[0059] Theoretically, peptides must be larger than approximately 1500 Da (approximately 15 amino acids) in size to bind to cell membrane-bound IgE, and larger than approximately 3000 Da (approximately 30 amino acids) in size to cross-link IgE molecules and induce an immune response (Nutten, 2018. EMJ Allergy Immunol, 3(1), pp.50-59).

[0060] Thus, preferably, at least about 95%, at least about 98%, at least about 99%, or about 100% by weight of the peptides have a molecular weight of less than about 3000 Da. There may be no detectable peptides of a size greater than or equal to about 3000 Da.

[0061] Thus, suitably, at least about 95%, at least about 98%, at least about 99%, or about 100% by weight of the peptides have a molecular weight of less than about 1500 Da. Preferably, at least 99% by weight of the peptides have a molecular weight of less than about 1500 Da. There may be no detectable peptides of a size greater than or equal to about 1500 Da.

[0062] Preferably, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% by weight of the peptides have a molecular weight of less than about 1200 Da. More preferably, at least 95% or 98% by weight of the peptides have a molecular weight of less than about 1200 Da.

[0063] Suitably, at least about 80%, at least about 85%, at least about 90%, or at least about 95% by weight of the peptides have a molecular weight of less than about 1000 Da. Preferably, at least about 95% by weight of the peptides have a molecular weight of less than about 1000 Da.

[0064] Preferably, the eHF of the present invention has no detectable peptides greater than about 3000 Da in size, and at least about 95% by weight of the peptides have a molecular weight of less than about 1200 Da.

[0065] A high proportion of dipeptides and tripeptides may improve nitrogen (protein) absorption in patients with intestinal dysfunction. PEPT1 is a dedicated transport pathway that facilitates the absorption of small peptides (e.g., dipeptides and tripeptides). In the first few weeks of life, intestinal PEPT1 is important for nutrient uptake and later for food transition after weaning.

[0066] Thus, at least about 30%, at least about 40%, or at least about 50% by weight of the peptides are dipeptides and tripeptides. Preferably, at least about 45%, at least about 50%, 45-55%, or 50-54% by weight of the peptides are dipeptides and tripeptides. More preferably, about 51-53%, or most preferably about 52% by weight of the peptides are dipeptides and tripeptides.

[0067] Suitably, at least about 30%, at least about 40%, or at least about 50% by weight of the peptides have a molecular weight of 240 to 600 Da. Preferably, at least about 45%, at least about 50%, 45 to 55%, or 50 to 54% by weight of the peptides have a molecular weight of 240 to 600 Da. More preferably, about 51 to 53%, or most preferably about 52% by weight of the peptides have a molecular weight of 240 to 600 Da.

[0068] The peptides in the eHF may have a median molecular weight of 300 Da to 370 Da, preferably 320 Da to 360 Da.

[0069] The major recognized allergens in cow's milk are α-lactalbumin (aLA), β-lactoglobulin (bLG), and bovine serum albumin (BSA).

[0070] Therefore, preferably, eHF can have an undetectable aLA content, for example, about 0.010 mg / kg or less of aLA, eHF can have an undetectable bLG content, for example, about 0.010 mg / kg or less of bLG, and / or eHF can have an undetectable BSA content, for example, about 0.010 mg / kg or less of BSA. Preferably, eHF of the present invention does not contain detectable amounts of aLA, bLG, and BSA. The contents of aLA, bLG, and BSA can be determined by any method known to those skilled in the art, for example, by ELISA.

[0071] Hydrolysis method Proteins for use in the eHF of the present invention can be hydrolyzed by any suitable method known in the art. For example, proteins can be enzymatically hydrolyzed using, for example, a protease. For example, proteins can be hydrolyzed using Alcalase (e.g., at an enzyme:substrate ratio of about 1 to 15% by weight for a duration of about 1 to 10 hours). The temperature can be in the range of about 40°C to 60°C, e.g., about 55°C. The reaction time can be, for example, 1 to 10 hours, and the pH value before starting the hydrolysis can be, for example, in the range of 6 to 9, preferably 6.5 to 8.5, and more preferably 7.0 to 8.0.

[0072] Porcine enzymes, particularly porcine pancreatic enzymes, may be used in the hydrolysis process. For example, WO 9304593(A1) discloses a hydrolysis process using trypsin and chymotrypsin. This process involves a two-step hydrolysis reaction, with a heat denaturation step between the steps to ensure that the final hydrolysate is substantially free of intact allergenic proteins. The trypsin and chymotrypsin used in these methods are preparations made from porcine pancreatic extracts.

[0073] WO2016156077A1 discloses a process for preparing a milk protein hydrolysate, which comprises hydrolyzing a milk-based proteinaceous material with a microbial alkaline serine protease in combination with bromelain, an Aspergillus-derived protease, and a Bacillus-derived protease.

[0074] Human milk oligosaccharides The eHF of the present invention may also contain one or more human milk oligosaccharides (HMOs).

[0075] It is known that HMOs can be found in high concentrations in human breast milk. They are highly resistant to enzymatic hydrolysis, suggesting that they may exhibit important functions unrelated to their caloric value. In particular, they have been shown to play essential roles in early infant development, such as immune system maturation. Many types of HMOs are found in human breast milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine, with a wide variety of linkages between them, resulting in a large number of different oligosaccharides in human breast milk, with over 130 such structures identified to date. Nearly all of these oligosaccharides have a lactose residue at the reducing end, with sialic acid and / or fucose (if present) occupying the non-reducing terminal positions. HMOs can be acidic (e.g., charged sialic acid-containing oligosaccharides) or neutral (e.g., fucosylated oligosaccharides).

[0076] Suitably, the eHF of the present invention may comprise at least one fucosylated oligosaccharide.

[0077] "Fucosylated oligosaccharides" are oligosaccharides containing fucose residues. Such oligosaccharides have neutral properties. One or several types of fucosylated oligosaccharides may be present. Fucosylated oligosaccharides include 2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, lacto-N-fucopentaose (lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, etc.), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose (fucosyllactose V, etc.). In some embodiments, the fucosylated oligosaccharide may be selected from the list comprising fucosyl lacto-N-neohexaose I, fucosyl lacto-N-neohexaose II, difucosyl lacto-N-hexaose I, difuco-lacto-N-neohexaose, difucosyl lacto-N-neohexaose I, difucosyl lacto-N-neohexaose II, fucosyl-para-lacto-N-hexaose, tri-fuco-para-lacto-N-hexaose I, and any combination thereof. In some embodiments, the fucosylated oligosaccharide comprises a 2'-fucosyl-epitope. The fucosylated oligosaccharide can be selected from the list including, for example, 2'-fucosyllactose, difucosyllactose, lacto-N-fucopentaose, lacto-N-fucohexaose, lacto-N-difucohexaose, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose, difuco-lacto-N-neohexaose, difucosyllacto-N-neohexaose, fucosyl-para-lacto-N-hexaose, and any combination thereof.

[0078] In a preferred embodiment, the eHF of the present invention comprises 2'-fucosyllactose. In some embodiments, there are no other types of fucosylated oligosaccharides than 2'-fucosyllactose, i.e., the eHF of the present invention comprises only 2'-fucosyllactose as a fucosylated oligosaccharide.

[0079] Suitably, the eHF of the present invention may comprise at least one N-acetylated oligosaccharide.

[0080] The term "N-acetylated oligosaccharides" encompasses both "N-acetyl-lactosamine" and "oligosaccharides containing N-acetyl-lactosamine." These are neutral oligosaccharides having N-acetyl-lactosamine residues. One or several types of N-acetylated oligosaccharides may be present. The N-acetylated oligosaccharides may be, for example, lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), or any combination thereof. In some embodiments, the N-acetylated oligosaccharides are lacto-N-neotetraose (LNnT), para-lacto-N-neohexaose (para-LNnH), or any combination thereof. In some embodiments, the N-acetylated oligosaccharides are LNnT. In some embodiments, the N-acetylated oligosaccharides are LNT. In some other embodiments, the N-acetylated oligosaccharides are a mixture of LNT and LNnT. In some embodiments, the eHF includes both LNT and LNnT in a ratio of LNT:LNnT of 5:1 to 1:2, or 2:1 to 1:1, or 2:1.2 to 2:1.6.

[0081] In a preferred embodiment, the eHF according to the present invention comprises lacto-N-neotetraose (LNnT). In some embodiments, no other N-acetylated oligosaccharides are present than lacto-N-neotetraose (LNnT), i.e., the eHF of the present invention comprises only lacto-N-neotetraose (LNnT) as an N-acetylated oligosaccharide.

[0082] N-acetylated oligosaccharides can be chemically synthesized by the enzymatic transfer of sugar units from a donor moiety to an acceptor moiety using glycosyltransferases, as described, for example, in U.S. Pat. No. 5,288,637 and WO 96 / 10086. Alternatively, LNTs and LNnTs can be prepared by chemically converting a ketohexose (e.g., fructose) that is free or bound to an oligosaccharide (e.g., lactulose) into an N-acetylhexosamine or an N-acetylhexosamine-containing oligosaccharide, as described in Wrodnigg, TM; Stutz, AE (1999) Angew. Chem. Int. Ed. 38:827-828. The N-acetyl-lactosamine thus generated can then be transferred to lactose as the acceptor moiety.

[0083] Preferably, the eHF of the present invention comprises at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide.

[0084] In some embodiments, the eHF of the present invention comprises an oligosaccharide mixture containing 2'-fucosyllactose (2-FL) and / or lacto-N-neotetraose (LNnT). In a preferred embodiment, the eHF of the present invention comprises an oligosaccharide mixture containing 2'-fucosyllactose (2-FL) and lacto-N-neotetraose (LNnT). The eHF of the present invention may contain only 2'-fucosyllactose (2-FL) as a fucosylated oligosaccharide and only lacto-N-neotetraose (LNnT) as an N-acetylated oligosaccharide.

[0085] Fucosylated oligosaccharides may be present in an eHF according to the present invention in a total amount of 0.5-3 g per liter of eHF, e.g., 0.8-1.5 g per liter (when formulated as directed). In some embodiments, the total amount of fucosylated oligosaccharides may be 0.85-1.3 g per liter of eHF, e.g., 0.9-1.25 g, 0.9-1.1 g, 1-1.25 g, or 1-1.2 g per liter (when formulated as directed). Preferably, the eHF (when formulated as directed) contains about 1 g / L of fucosylated oligosaccharides, e.g., about 1 g / L of 2'-fucosyllactose.

[0086] N-acetylated oligosaccharides may be present in eHFs according to the present invention in a total amount of 0.2-1 g per liter of eHF, e.g., 0.5-0.8 g per liter (when formulated as directed). In some embodiments, the total amount of N-acetylated oligosaccharides may be 0.5-0.75 g, 0.5-0.7 g, or 0.5-0.6 g per liter of eHF (when formulated as directed). Preferably, the eHF (when formulated as directed) contains about 0.5 g / L of N-acetylated oligosaccharides, e.g., about 0.5 g / L of lacto-N-neotetraose.

[0087] All of these different ranges can be combined together.

[0088] Thus, in one embodiment of the present invention, eHF (when formulated as directed) comprises at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide (e.g., 2'-fucosyllactose and lacto-N-neotetraose); (i) the total amount of fucosylated oligosaccharides is 0.8 to 1.5 g per liter of eHF; and / or (ii) The total amount of N-acetylated oligosaccharides is 0.5 to 0.8 g per 1 L of eHF.

[0089] In another embodiment, the eHF of the present invention (when formulated as directed) comprises at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide (e.g., 2'-fucosyllactose and lacto-N-neotetraose); (i) the total amount of fucosylated oligosaccharides is between 0.9 and 1.25 g per liter of eHF; and / or (ii) The total amount of N-acetylated oligosaccharides is 0.5 to 0.7 g per 1 L of eHF.

[0090] In another embodiment, the eHF of the present invention (when formulated as directed) comprises at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide (e.g., 2'-fucosyllactose and lacto-N-neotetraose); (i) the total amount of fucosylated oligosaccharides is 1 to 1.2 g per liter of eHF; and / or (ii) The total amount of N-acetylated oligosaccharides is 0.5 to 0.6 g per 1 L of eHF.

[0091] In a preferred embodiment, the eHF of the present invention (when formulated as directed) contains about 1 g / L of fucosylated oligosaccharides and about 0.5 g / L of N-acetylated oligosaccharides. In a more preferred embodiment, the eHF of the present invention (when formulated as directed) contains about 1 g / L of 2'-fucosyllactose and about 0.5 g / L of lacto-N-neotetraose.

[0092] The eHF of the present invention may contain 0.075-0.5 g / 100 kcal, 0.1-0.3 g / 100 kcal, or 0.12-0.25 g / 100 kcal of 2'-fucosyllactose and about 0.03-0.15 g / 100 kcal, 0.05-0.12 g / 100 kcal, or 0.05-0.1 g / 100 kcal of lacto-N-neotetraose. Preferably, the eHF of the present invention contains about 0.15 g / 100 kcal of 2'-fucosyllactose and about 0.075 g / 100 kcal of lacto-N-neotetraose.

[0093] The fucosylated and N-acetylated oligosaccharides contained in the eHF according to the invention are typically present in a ratio of fucosylated to N-acetylated oligosaccharides of 2.0:0.54 to 2.0:2.26, for example, 2.0:0.76 to 2.0:1.8 or 2.0:0.8 to 2.0:1.4. In a particularly advantageous embodiment, this ratio is 2.0:1 or about 2.0:1.

[0094] In a preferred embodiment, the eHF of the present invention contains about 1 g / L 2'-fucosyllactose and about 0.5 g / L lacto-N-neotetraose, and / or about 0.15 g / 100 kcal 2'-fucosyllactose and about 0.075 g / 100 kcal lacto-N-neotetraose; about 0 wt% of the lipids are MCTs, and / or the eHF does not contain added MCTs; optionally, free amino acids are present at a concentration of 20-25 wt%, 21-23 wt%, or about 22 wt%, based on the total weight of amino acids.

[0095] In a preferred embodiment, the eHF of the present invention has no detectable peptides greater than about 3000 Da in size, and at least about 95% by weight of the peptides have a molecular weight of less than about 1200 Da; optionally, at least about 45%, at least about 50%, or 45-55% by weight of the peptides have a molecular weight of 240-600 Da, and / or the free amino acids are present at a concentration of 20-25%, 21-23%, or about 22% by weight based on the total weight of amino acids; about 0% by weight of the lipids are MCTs; the eHF of the present invention contains about 1 g / L 2'-fucosyllactose and about 0.5 g / L lacto-N-neotetraose, and / or about 0.15 g / 100 kcal 2'-fucosyllactose and about 0.075 g / 100 kcal lacto-N-neotetraose.

[0096] Free amino acids The eHF of the present invention may contain free amino acids.

[0097] Free amino acids can be incorporated into the eHF of the present invention to supplement the amino acids contained in the peptide. The level of free amino acids can be selected to provide an amino acid profile sufficient for infant nutrition, particularly an amino acid profile that meets nutritional regulations (e.g., European Commission Directive 2006 / 141 / EC).

[0098] Examples of free amino acids for use in the eHF of the present invention include histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, carnitine, taurine, and mixtures thereof.

[0099] Free amino acids provide an equivalent source of protein (i.e., contribute to the nitrogen content). As mentioned above, a high proportion of dipeptides and tripeptides can improve nitrogen (protein) absorption in patients with intestinal dysfunction. Therefore, a low proportion of free amino acids can improve nitrogen (protein) absorption in patients with intestinal dysfunction.

[0100] Thus, suitably, the free amino acids may be present at a concentration of 50% by weight or less, 40% by weight or less, 30% by weight or less, or 25% by weight or less, based on the total weight of the amino acids. Preferably, the eHF contains 25% by weight or less of free amino acids, based on the total weight of the amino acids.

[0101] More preferably, the free amino acids are present at a concentration of 20-25 wt %, 21-23 wt %, or about 22 wt %, based on the total weight of the amino acids.

[0102] The free amino acid content can be determined by any method known to those skilled in the art. Preferably, the free amino acid content can be obtained by separating the free amino groups present in the aqueous sample extract by ion exchange chromatography and optical detection after post-column derivatization with ninhydrin reagent. The total amino acid content can be obtained by hydrolysis of the test portion in 6 mol / L HCl under nitrogen and separating the individual amino acids by ion exchange chromatography as described above.

[0103] carbohydrates The carbohydrate content of the eHF of the present invention is preferably in the range of 9-14 g of carbohydrate per 100 kcal.

[0104] The carbohydrate may be any carbohydrate suitable for use in lipid eHF.

[0105] Examples of carbohydrates for use in the eHF of the present invention include lactose, sucrose, maltodextrin, and starch. Mixtures of carbohydrates may also be used.

[0106] In one embodiment, the carbohydrate content comprises maltodextrin, hi one embodiment, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% by weight of the total carbohydrate content is maltodextrin.

[0107] In one embodiment, the carbohydrate content comprises lactose, hi one embodiment, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% by weight of the total carbohydrate content is lactose.

[0108] In one embodiment, the carbohydrates include lactose and maltodextrin.

[0109] lipids The lipid content of the eHF of the present invention is preferably in the range of 4.0 to 6.0 g of lipid per 100 kcal.

[0110] The lipid can be any lipid or lipid suitable for use in eHF.

[0111] Examples of lipids for use in the eHF of the present invention include sunflower oil, low-erucic acid rapeseed oil, safflower oil, canola oil, olive oil, coconut oil, palm kernel oil, soybean oil, fish oil, palm olein, high-oleic sunflower oil, and high-oleic safflower oil, and microbial fermentation oils containing long-chain polyunsaturated fatty acids.

[0112] Lipids can also be in the form of oil-derived fractions such as palm olein, medium-chain triglycerides (MCTs), and esters of fatty acids (e.g., arachidonic acid, linoleic acid, palmitic acid, stearic acid, docosahexaenoic acid, linolenic acid, oleic acid, lauric acid, capric acid, caprylic acid, caproic acid, etc.).

[0113] Further examples of lipids include structured lipids (i.e., lipids that have been chemically or enzymatically modified to alter their structure). Preferably, the structured lipids are sn2 structured lipids, such as triglycerides that have an increased proportion of palmitic acid at the sn2 position of the triglyceride. Structured lipids may or may not be included.

[0114] Oils such as fish or microbial oils that are rich in preformed arachidonic acid (ARA) and / or docosahexaenoic acid (DHA) may also be added.

[0115] Long chain polyunsaturated fatty acids such as dihomo-γ-linolenic acid, arachidonic acid (ARA), eicosapentaenoic acid, and docosahexaenoic acid (DHA) may also be added.

[0116] The eHF may contain 2-20 mg ARA per 100 kcal, 5-15 mg ARA per 100 kcal, or about 10 mg ARA per 100 kcal, and / or 2-20 mg DHA per 100 kcal, 5-15 mg DHA per 100 kcal, or about 10 mg DHA per 100 kcal. Preferably, the eHF contains about 10 mg ARA per 100 kcal and about 10 mg DHA per 100 kcal.

[0117] Medium Chain Triglycerides (MCTs) High concentrations of MCTs may impair early weight gain. MCTs are not stored and do not support lipid accumulation. For example, Borschel et al. reported that infants fed a formula containing no MCTs gained significantly more weight between days 1 and 56 than infants fed a formula containing 50% MCT-derived lipids (Borschel, M., et al., 2018. Nutrients, 10(3), p. 289).

[0118] Thus, in the eHF of the present invention, no more than about 30% by weight of the lipids are medium chain triglycerides (MCTs).

[0119] In some embodiments, about 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, 0.5% by weight or less, or 0.1% by weight or less of the lipids are medium chain triglycerides (MCTs).

[0120] In some embodiments, 0-30%, 0-25%, 0-20%, 0-15%, 0-10%, 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 0-0.5%, or 0-0.1% by weight of the lipids are medium chain triglycerides (MCTs).

[0121] Preferably, the eHF does not contain added MCTs. Preferably, about 0% by weight of the lipids are MCTs and / or the eHF does not contain detectable MCTs. Preferably, the eHF does not contain MCTs.

[0122] In a preferred embodiment, the eHF of the present invention has no detectable peptides greater than or equal to about 3000 Da in size; at least about 95% by weight of the peptides have a molecular weight of less than about 1200 Da; 45-55% by weight of the peptides have a molecular weight of 240-600 Da; free amino acids are present at a concentration of 20-25% by weight based on the total weight of amino acids; and the eHF does not contain added MCTs.

[0123] Further ingredients The eHF of the present invention also preferably contains nutritionally significant amounts of all vitamins and minerals considered essential in the daily diet, with minimum requirements established for certain vitamins and minerals.

[0124] Examples of vitamins, minerals, and other nutrients for use in the eHF of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in the form of their salts.

[0125] The eHF of the present invention may include one or more carotenoids.

[0126] The eHF of the present invention may also contain at least one probiotic. The term "probiotic" refers to a preparation of microbial cells or components of microbial cells that have a beneficial effect on the health or well-being of the host. Specifically, probiotics can improve intestinal barrier function.

[0127] Preferred probiotics are generally safe, L(+) lactic acid producing cultures, and have an acceptable shelf life for products that are required to remain stable and effective for up to 24 months.

[0128] Examples of probiotic microorganisms for use in the eHF of the present invention include yeasts such as Saccharomyces, Debaromyces, Candida, Pichia, and Torulopsis, as well as bacteria of the genera Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, and the like. Examples of bacteria that may be present include bacteria of the genera Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus, and Lactobacillus.

[0129] Specific examples of suitable probiotic microorganisms are Saccharomyces cerevisiae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei subsp. casei, Lactobacillus casei shirota, Lactobacillus curvatus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus casei ... Lactobacillus falciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus rhamnosus (Lactobacillus GG), Lactobacillus salmonis, Lactobacillus lactis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosus, Pediococcus acidilactici, Pediococcus halophilus, Streptococcus faecalis, Streptococcus thermophilus, Staphylococcus carnosis, and Staphylococcus xylosus.

[0130] The eHF of the present invention may also contain other substances that may have beneficial effects, such as prebiotics, lactoferrin, dietary fiber, nucleotides, nucleosides, etc.

[0131] Treatment method In one aspect, the invention provides a method of feeding an infant, comprising administering to the infant an eHF according to the invention.

[0132] The term "infant" refers to a child under 12 months of age, for example, a child between 0 and 6 months of age. An infant is a human.

[0133] Milk protein allergy Preferably, the infant has a cow's milk protein allergy. The term "allergy" refers to a hypersensitivity of the immune system to a substance that is normally tolerated. The allergy may be a doctor-diagnosed allergy.

[0134] Cow's milk proteins (CMPs) are a major cause of food allergies in infants, affecting 2-3% of children worldwide. Most children with CMP allergies (CMPA) have two or more symptoms: 50-70% have skin symptoms; 50-60% have gastrointestinal symptoms; and 20-30% have respiratory tract symptoms. 10% of children may develop severe, life-threatening symptoms. (Nutten, 2018. EMJ Allergy Immunol, 3(1), pp. 50-59)

[0135] Skin symptoms may include urticaria, atopic eczema, and angioedema. Gastrointestinal symptoms may include dysphagia, frequent regurgitation, colic, abdominal pain, vomiting, anorexia, anorexia, diarrhea (with or without intestinal protein loss or bleeding), constipation (with or without perianal rash), failure to thrive, occult blood loss, and iron deficiency anemia. Respiratory symptoms may include runny nose, wheezing, and chronic cough. Other systemic symptoms include anaphylaxis and shock-like symptoms with severe metabolic acidosis, vomiting, and diarrhea (food protein-induced gastroenteritis). These symptoms are typically unrelated to infection, drug ingestion, or other causes (Koletzko, S., et al., 2012. Journal of pediatric gastroenterology and nutrition, 55(2), pp. 221-229).

[0136] If breastfeeding is not possible, specialized infant formulas are recommended. ESPGHAN recommends the use of extensively hydrolyzed protein-based formulas, or eHF, for non-breastfed infants with CMPA, which have been proven effective for infants with CMPA (Koletzko, S., et al., 2012. Journal of pediatric gastroenterology and nutrition, 55(2), pp. 221-229).

[0137] Thus, in one aspect the present invention provides a method of treating and / or preventing cow's milk protein allergy and / or one or more of urticaria, atopic eczema, angioedema, dysphagia, frequent regurgitation, colic, abdominal pain, vomiting, loss of appetite, refusal to eat, diarrhea (with or without intestinal protein loss or bleeding), constipation (with or without perianal rash), failure to thrive, occult blood, iron deficiency anaemia, runny nose, wheezing, chronic cough, anaphylaxis, and shock-like symptoms associated with severe metabolic acidosis, vomiting, and diarrhea (food protein induced gastroenteritis) in an infant, preferably wherein the infant is free of infection, the method comprising administering to the infant an eHF according to the present invention.

[0138] In another aspect, the present invention provides an eHF according to the present invention for use in the treatment and / or prevention of cow's milk protein allergy and / or one or more of urticaria, atopic eczema, angioedema, dysphagia, frequent regurgitation, colic, abdominal pain, vomiting, loss of appetite, refusal to eat, diarrhea (with or without intestinal protein loss or bleeding), constipation (with or without perianal rash), failure to thrive, occult blood, iron deficiency anaemia, runny nose, wheezing, chronic cough, anaphylaxis, and shock-like symptoms associated with severe metabolic acidosis, vomiting, and diarrhea (food protein induced gastroenteritis) in an infant, preferably wherein the infant is free of infection.

[0139] obesity Infants with CMPA may require higher levels of protein due to gastrointestinal symptoms, for example. Therefore, eHF typically contains 2.6–2.8 g of protein per 100 kcal to accommodate the needs of infants with gastrointestinal pathologies, including severe malabsorption, or who require more protein and calories to compensate for a high metabolic rate.

[0140] However, consumption of high-protein infant formula has been associated with increased body weight and body mass index at age 2 years, as well as elevated plasma concentrations of essential amino acids, insulin-like growth factor-1, and C-peptide, which may induce weight gain and lipogenic activity. Lower protein content may reduce the risk of subsequent obesity (Totzauer, M., et al., 2018. Obesity, 26(7), pp. 1203-1210).

[0141] For example, the European Childhood Obesity Project (CHOP) has shown that infants fed infant formulas containing high protein levels gain more weight during the first year of life and have a higher BMI and risk of obesity at age 6 than infants fed infant formulas containing lower protein levels (Totzauer, M., et al., 2018. Obesity, 26(7), pp.1203-1210).

[0142] Importantly, eHF should still be suitable for infants with CMPA, however, the reduced amount of protein may not support proper growth and development in allergic infants.

[0143] The inventors have surprisingly shown that the eHF of the present invention supported proper growth and development in allergic infants. Moreover, eHF was safe and well tolerated.

[0144] Thus, in one aspect, the present invention provides a method for preventing obesity in an infant and / or reducing the risk of obesity in an infant, the method comprising administering an eHF according to the present invention to the infant. Preferably, the infant has a cow's milk protein allergy. The method may also treat and / or prevent cow's milk protein allergy in the infant.

[0145] In another aspect, the present invention provides an eHF according to the present invention for use in preventing obesity in an infant and / or reducing the risk of obesity in an infant. Preferably, the infant has a cow's milk protein allergy. In particular, the present invention provides an eHF according to the present invention for use in preventing obesity in an infant and / or reducing the risk of obesity in an infant; and for use in treating and / or preventing cow's milk protein allergy (CMPA) and / or one or more symptoms of CMPA.

[0146] Manufacturing method The eHF of the present invention can be prepared by any suitable method.

[0147] For example, eHF can be prepared by blending together a hydrolyzed protein source, a carbohydrate source, and a lipid source in appropriate proportions. If an emulsifier is used, it can be added at this point. Vitamins and minerals can also be added at this point, although vitamins are typically added later to avoid thermal degradation. Any lipophilic vitamins and emulsifiers can be dissolved in the lipid source before blending. Water, preferably water treated with reverse osmosis, can then be mixed to form a liquid mixture. A commercially available liquefaction device can be used to form the liquid mixture. The liquid mixture can then be homogenized.

[0148] The liquid mixture can then be heat treated to reduce the bacterial content, for example by steam injection or using an autoclave or a heat exchanger, for example a plate heat exchanger.

[0149] The liquid mixture may then be cooled and / or homogenized. The pH and solids content of the homogenized mixture may be adjusted at this point.

[0150] The homogenized mixture can then be transferred to a suitable drying device, such as a spray dryer or freeze dryer, and converted to a powder. If liquid eHF is preferred, the homogenized mixture can be sterilized and then aseptically filled into suitable containers, or it can be filled into containers first and then retorted. [Example]

[0151] The present invention will now be further described by way of examples, which are meant to be provided to aid those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way.

[0152] Example 1 - Exemplary Highly Hydrolyzed Infant Formula The following is an exemplary extensively hydrolyzed infant formula according to the present invention. The eHF of the present invention preferably contains nutritionally significant amounts of all nutrients, vitamins, and minerals considered essential in the daily diet. Minimum requirements have been established for certain nutrients, vitamins, and minerals.

[0153] [Table 1]

[0154] Example 2 - Safety and efficacy of a highly hydrolyzed infant formula with reduced protein content. Study design The safety and efficacy of a reduced-protein, extensively hydrolyzed infant formula were investigated in a controlled, double-blind, randomized, multicenter, interventional clinical trial with two parallel groups fed the formula.

[0155] The primary objective of this clinical trial was to demonstrate that infants with cow's milk protein allergy (CMPA) fed a novel eHF (test formula) with reduced protein levels (Althera 2.2) and two added human milk oligosaccharides (HMOs) performed equally well as infants fed a commercially available eHF (Althera 2.5) (control formula) without HMOs. Commercial eHF is currently approved as a food for specific medical purposes (Regulation (EU) 2016 / 128). The primary endpoint of the study was daily weight gain from enrollment to the 4-month follow-up, with the age range at enrollment being 0-6 months (non-inferiority design).

[0156] Secondary objectives included whether ingestion of the test formula by CMPA infants was well tolerated and enabled age-appropriate growth. Therefore, secondary study endpoints included changes in weight-for-age, height-for-age, and other growth parameters, including head circumference Z-score (WHO growth standards).

[0157] The study population consisted of full-term infants with physician-diagnosed CMPA according to standard clinical practice and who met at least two of the inclusion criteria. A total of 130 infants were required to complete 4 months of study formula intake.

[0158] The selection criteria were as follows: 1. Full-term infants (37 weeks ≤ gestational age ≤ 42 weeks); 2.2500g≦birth weight≦4500g; 3. Written informed consent from the infant's parent (or both parents, if required by national regulations) or legal representative (LAR); 4. Infants up to 6 months of age; 5. Those who are formula-fed exclusively at the time of enrollment or mothers of breastfed CMPA infants who voluntarily chose to feed formula exclusively before enrollment: and 6. Infants diagnosed with CMPA by a physician according to standard clinical practice (and not being treated with extensively hydrolyzed milk or amino acid infant formula) who have the presence of at least two of the following symptoms: crying, regurgitation, liquid stools or constipation, atopic skin lesions, urticaria, or respiratory symptoms. For a diagnosis based on either a positive IgE blood test, skin prick test, patch test, or food challenge, only one of the above symptoms must be present.

[0159] The exclusion criteria were as follows: 1. Congenital diseases or birth defects that may affect development. 2. Chronic malabsorption documented not to be due to CMPA. 3. Significant prenatal and / or postnatal serious illness other than CMPA prior to enrollment (as determined by the investigator's medical judgment). 4. Parents of a minor. 5. Infants whose parents or caregivers cannot be expected to comply with study procedures. 6.Currently participating in another clinical trial or have participated in one since birth.

[0160] The test and control formulas are shown below.

[0161] [Table 2]

[0162] In the test and control formulas, Over 99% by weight of the peptides had a molecular weight of less than 3000 Da. Over 95% by weight of the peptides had a molecular weight less than 1200 Da. Approximately 52% by weight of the peptides were dipeptides and tripeptides (peptides with molecular weights of 600 to 240 Da). Approximately 22.4% by weight of the total amino acids were free amino acids in the test and control formulas. There was no detectable beta-lactoglobulin (i.e., the beta-lactoglobulin content was less than 0.01 mg / kg). There was no detectable casein (i.e., the casein content was less than 0.2 mg / kg).

[0163] Both formulas were in powder form and were prepared for oral administration by the infant in amounts appropriate for their weight, age, and appetite, according to the instructions printed on the product label on the can.

[0164] Infants received the study formula until a minimum of 4 months after baseline (main study period) and for as long as the infant needed according to medical prescription (up to 12 months of age).

[0165] The daily intake required by the infants varied according to their age, weight, and appetite. The products were given to the infants ad libitum, but parents or caregivers followed the guidelines printed on the label and / or received advice from study personnel regarding the appropriate daily intake.

[0166] Infants will have up to seven study visits: baseline (at enrollment), monthly (months +1, +2, +3, +4) from baseline until 4 months after baseline, and 6 months after baseline. One additional final visit is planned when the infant reaches 12 months of age.

[0167] Randomization was 1:1 per study formula group and was performed by minimization in Medidata Balance. Stratification was by age at enrollment (0-60 days, 61-120 days, >120 days), sex, and mode of delivery (vaginal or cesarean section). Twins enrolled were randomized to the same formula.

[0168] Test results The study showed that the test formula supported proper growth and development in allergic infants and was safe and well tolerated.

[0169] The primary analysis assessed whether growth was noninferior for infants fed the test formula compared with infants fed the control formula over the first 4 months of treatment. Treatment differences and one-sided simultaneous 97.5% confidence intervals were calculated using mixed models. Weight (kg) was modeled as a function of age (months), treatment, sex, age * treatment, and age * sex. The model assumed a variance-covariance matrix with an autocorrelated type I structure for outcomes at adjacent visits. The calculated difference was used to estimate the treatment effect at day 63 [(14 + 112) / 2]. Estimates were converted from kg / month to g / day using a divisor of 0.0305. Key secondary analyses included weight, length, and head circumference.

[0170] Infants fed the test formula with reduced protein and HMO content achieved normal growth in line with WHO growth standards (Figures 1-6). Specifically, the primary analysis showed that weight gain [g / d] of infants fed the test formula was noninferior to growth on the control formula (Figures 1-3). There were no significant differences in any of the anthropometric parameters at any time point up to the 4-month follow-up (Figures 4-6).

[0171] The safety profiles of the test and control formulas were similar: there were no significant between-group differences in the incidence of adverse events of interest.

[0172] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the disclosed methods, cells, compositions, and uses of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention, which are obvious to those skilled in the art, are intended to be within the scope of the following claims.

Claims

1. 1. An extensively hydrolyzed infant formula (eHF) comprising protein, carbohydrates, and lipids, wherein the eHF contains about 2.4 g or less of protein per 100 kcal, and about 30% or less by weight of the lipids are medium chain triglycerides (MCTs).

2. The eHF of claim 1, wherein the eHF comprises 2'-fucosyllactose (2'FL) and / or lacto-N-neotetraose (LNnT), preferably the eHF comprises 2'FL and LNnT.

3. 3. The eHF of claim 1 or claim 2, wherein the eHF comprises 0.5 to 3 g / L, 0.8 to 1.5 g / L, or about 1 g / L of 2'FL, preferably about 1 g / L of 2'FL.

4. 4. The eHF of any one of claims 1 to 3, wherein the eHF comprises 0.2 to 1 g / L, 0.5 to 0.8 g / L, or about 0.5 g / L of LNnT, preferably, the eHF comprises about 0.5 g / L of LNnT.

5. The eHF of any one of claims 1 to 4, wherein the eHF contains about 1.8 g or more of protein per 100 kcal.

6. 6. The eHF of any one of claims 1 to 5, wherein the eHF comprises 1.8 to 2.4 g of protein per 100 kcal, 2.1 to 2.3 g of protein per 100 kcal, or 2.15 to 2.25 g of protein per 100 kcal.

7. The eHF of any one of claims 1 to 6, wherein the eHF contains about 2.2 g of protein per 100 kcal.

8. 8. The eHF of any one of claims 1 to 7, wherein about 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, 5% by weight or less, or 1% by weight or less of the lipids are medium chain triglycerides (MCTs).

9. The eHF of any one of claims 1 to 8, wherein the eHF does not contain added MCT.

10. 10. The eHF of any one of claims 1 to 9, wherein at least about 95%, at least about 98%, at least about 99%, or about 100% by weight of the peptides in the eHF have a molecular weight of less than about 3000 Da, and preferably there are no detectable peptides in the eHF that are greater than or equal to about 3000 Da in size.

11. 11. The eHF of any one of claims 1 to 10, wherein at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% by weight of the peptides in the eHF have a molecular weight of less than about 1200 Da, preferably at least about 98% by weight of the peptides in the eHF have a molecular weight of less than about 1200 Da.

12. 12. The eHF of any one of claims 1 to 11, wherein at least about 45 wt%, at least about 50 wt%, 45-55 wt%, or 50-54 wt% of the peptides in the eHF are dipeptides and tripeptides, preferably 51-53 wt% of the peptides in the eHF are dipeptides and tripeptides, or more preferably about 52 wt% of the peptides in the eHF are dipeptides and tripeptides.

13. 13. The eHF of any one of claims 1 to 12, wherein at least about 45%, at least about 50%, 45-55%, or 50-54% by weight of the peptides in the eHF have a molecular weight of 240-600 Da, preferably 51-53% by weight of the peptides in the eHF have a molecular weight of 240-600 Da, more preferably about 52% by weight of the peptides in the eHF have a molecular weight of 240-600 Da.

14. 14. The eHF of any one of claims 1 to 13, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the protein is whey protein.

15. 15. The eHF of any one of claims 1 to 14, wherein the eHF comprises free amino acids, preferably the free amino acids are present at a concentration of 50% by weight or less, 40% by weight or less, 30% by weight or less, or 25% by weight or less, based on the total weight of amino acids.

16. 16. The eHF of claim 15, wherein the free amino acids are present at a concentration of 20-25%, 21-23%, or about 22% by weight based on the total weight of amino acids.

17. 17. The eHF of any one of claims 1 to 16, wherein the eHF comprises 9 to 14 g of carbohydrates per 100 kcal and / or 4.0 to 6.0 g of lipids per 100 kcal.

18. 18. The eHF of any one of claims 1 to 17, wherein the eHF has an energy density of 60 to 72 kcal per 100 mL.

19. 20. A method of feeding an infant, comprising administering to said infant an eHF according to any one of claims 1 to 18, wherein said infant has a cow's milk protein allergy.

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